Intracellular delivery of peptides via association with ubiquitin or SUMO-1 coupled to protein transduction domains

Anne-Laure Vitte,Pierre Jalinot

doi:10.1186/1472-6750-8-24

Abstract

BackgroundWe previously developed small hybrid proteins consisting of SUMO-1 linked to an heptapeptide fused to the Tat protein transduction domain (PTD). The heptapeptide motif was selected from a library of random sequences to specifically bind HIV-1 regulatory proteins Tat or Rev. These constructs, named SHP, are able to enter primary lymphocytes and some of them inhibit HIV-1 replication. Considering these positive results and other data from the literature, we further tested the ability of ubiquitin or SUMO-1 linked to various PTD at their N-terminus to deliver within cells proteins or peptides fused downstream of their diglycine motif. In this system it is expected that the intracellular ubiquitin or SUMO-1 hydrolases cleave the PTD-Ub or PTD-SUMO-1 modules from the cargo polypeptide, thereby allowing its delivery under an unmodified form.ResultsSeveral bacterial expression vectors have been constructed to produce modular proteins containing from the N- to the C-terminus: the FLAG epitope, a cleavage site for a protease, a PTD, human ubiquitin or SUMO-1, and either GFP or the HA epitope. Nine different PTDs were tested, including the Tat basic domain, wild type or with various mutations, and stretches of arginine or lysine. It was observed that some of these PTDs, mainly the Tat PTD and seven or nine residues long polyarginine motifs, caused association of the hybrid proteins with cells, but none of these constructs were delivered to the cytosol. This conclusion was derived from biochemical and immunofluorescence studies, and also from the fact that free cargo protein resulting from cleavage by proteases after ubiquitin or SUMO-1 was never observed. However, in agreement with our previous observations, mutation of the diglycine motif into alanine-arginine, as in the SHP constructs, allows cytosol entry demonstrated by immunofluorescence observations on living cells and by cell fractionation analyses. This process results from a non-endocytic pathway.ConclusionOur observations indicate that fusion of SUMO-1 to a peptide-PTD module allows generation of a stable hybrid protein that is easily produced in bacteria and which efficiently enters into cells but this property necessitates mutation of the diglycine motif at the end of SUMO-1, thereby impairing delivery of the peptide alone.

Highlights

We previously developed small hybrid proteins consisting of SUMO-1 linked to an heptapeptide fused to the Tat protein transduction domain (PTD)
We tested a system which associates the protein with a module composed of the FLAG epitope, a PTD and either ubiquitin or SUMO-1 (Figure 1a)
The diglycine motif of these small proteins is directly linked to the first amino acid of the protein to be delivered, which in our constructions was green fluorescent protein (GFP), taken as a model polypeptide

Summary

Introduction

We previously developed small hybrid proteins consisting of SUMO-1 linked to an heptapeptide fused to the Tat protein transduction domain (PTD). These constructs, named SHP, are able to enter primary lymphocytes and some of them inhibit HIV-1 replication Considering these positive results and other data from the literature, we further tested the ability of ubiquitin or SUMO-1 linked to various PTD at their N-terminus to deliver within cells proteins or peptides fused downstream of their diglycine motif. Characterization of the capacity of these proteins to enter cells led to the mapping of peptidic domains of limited size responsible for this property which turned out to be transferable by linkage to various peptides or proteins [4,5,6]; for a recent review see [7]) These so-called protein transduction domains (PTD) or cell-penetrating peptides raised the possibility of delivering an exogenous protein component into cells.

Methods

Results

Conclusion